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526 Biophysical Journal Volume 93 July 2007 526–538

Rapid Membrane Fusion of Individual Particles with Supported Lipid Bilayers

Laura Wessels, Mary Williard Elting, Dominic Scimeca, and Keith Weninger Department of Physics, North Carolina State University, Raleigh, North Carolina

ABSTRACT Many enveloped employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus , and buffer conditions in membrane fusion.

INTRODUCTION The boundaries of all living cells and their compartments are fully mix both the lipids and the contents of fusing structures defined by lipid bilayers. Intracellular transport, cell entry, (25,26). and secretion require vesicular lipid structures to fuse with As valuable as these bulk assays have been in advancing target membranes. Membrane fusion proteins are involved in our understanding of membrane fusion, they have limitations. catalyzing almost every situation of biological membrane Low-pH triggered viral membrane fusion occurs rapidly fusion. Despite decades of intense study, the precise molec- after acidification, with timescales measured in tens of ular mechanism by which fusion proteins mediate membrane seconds or minutes in the bulk liposome experiments(16,21). fusion remains a subject of much debate (1–11). The stochastic nature of each occurrence of fusion prevents Among the best-studied membrane fusion machines the precise synchronization of all of the individual virus- are those present in enveloped viruses. Enveloped viruses liposome fusion events within a cuvette-based bulk solution have evolved highly efficient fusion proteins that allow the assay. These experimental challenges mask transient, inter- viral genome to penetrate targeted cells (12–18). For most mediate states along the fusion pathway and obscure detailed enveloped viruses, environmental signals trigger these pro- analysis of the trajectory of the nonequilibrium processes teins to catalyze fusion of the viral membrane with the cell driving the dynamics. Difficulty separating the linked pro- membrane. The decreased pH encountered along the endo- cesses of binding and fusion in bulk assays also complicates cytotic pathway is a common trigger for the fusion proteins the interpretation of experimental results. of many enveloped viruses, most notably influenza (19,20). We have developed a fluorescence assay to make detailed In a few cases, atomic resolution structures are available for measurements of the early stages of individual Sindbis and viral fusion proteins under both neutral pH and low pH con- influenza virus particles fusing to supported lipid bilayers ditions (12) that have led to formulation of models of their under acidic conditions. Our assay detects lipid mixing of molecular action (12,13). octadecyl rhodamine (R18) between virus particles and the Many enveloped viruses will fuse to protein free lipid supported lipid bilayer. As R18 is known to ‘‘flip-flop’’ be- bilayers (20–22). In vitro liposome fusion experiments have tween the inner and outer leaflets of a labeled bilayer (27,28), been extremely useful in characterizing the biochemical this signal does not differentiate between hemifusion and full properties of viral fusion, including the pH and lipid species fusion. Throughout this article we will use hemifusion/fusion dependences (23,24). Bulk liposome measurements have to indicate this ambiguity. also confirmed that viral fusion is often nonleaky and can The supported lipid bilayer geometry is desirable for its compatibility with high-resolution optical measurements as well as its ease of integration with biotechnological instru- mentation and sensors. Supported bilayers have been suc- Submitted September 14, 2006, and accepted for publication March 8, 2007. cessfully applied in membrane fusion studies of SNARE Address reprint requests to Keith Weninger, Dept. of Physics, North Carolina State University, Raleigh, NC 27695. E-mail: keith_weninger@ proteins (29–31) and influenza virus (32–36). Illumination of ncsu.edu. the supported lipid bilayer by total internal refection yields a Editor: Lukas K. Tamm. Ó 2007 by the Biophysical Society 0006-3495/07/07/526/13 $2.00 doi: 10.1529/biophysj.106.097485 Single Virus Bilayer Fusion 527 signal/noise ratio sufficient for precise measurements of (HBS, 45 mM Hepes, pH 7.4, 100 mM NaCl) (for influenza). We estimate individual virus particles during fusion. Continued develop- that gel filtration in NAP 5 columns diluted the samples to ;E the starting 3 12 ment of this single-particle approach will allow detection of concentration (0.7 10 particles/ml (Sindbis) or 0.07 or 0.7 mg/ml (influenza)). the transient, stochastic intermediate states that are commonly averaged in liposome fusion assays. We have verified that our supported bilayer assay repro- Lipids, flow cells, bilayers, and buffer exchanger duces the general trends seen in the bulk liposome exper- All lipids (including total liver extract, brain phosphatidylethanolamine iments for these viruses (16,21,37). Although preexposure to (PE), egg phosphatidylcholine (PC), brain sphingomyelin, and cholesterol) low pH for several minutes inactivates the virus for fusion were purchased from Avanti Polar Lipids (Alabaster, AL). Chloroform was (21) and infection (38), our results show that virus exposed removed from solutions of lipids and cholesterol (mixed at ratios indicated in the text) under flowing argon leaving a film on the surface of a glass tube. to low pH immediately before encountering the target mem- The lipid films were placed in vacuum for at least 2 h and then hydrated with brane maintains fusion capacity. Observation of individual Tris-buffered saline (TBS, 25 mM Tris-HCL, pH 7.3, 150 mM NaCl) (51). virus particles indicates that lipid mixing follows binding Small unilamellar vesicles were prepared by extrusion through 100-nm-pore rapidly, in under a half second for most conditions, and that filters (miniextruder, Avanti Polar Lipids) at 5 mg/ml (liver extract), 4 mg/ml the dynamics of lipid mixing in individual fusion events is (mixes), or 20 mg/ml (PC). Quartz microscope slides were cleaned with a sequence of bath sonication faster than our instrumental resolution limit of 30 ms. steps (soapy water, acetone, ethanol, 1 M potassium hydroxide, and water), Cholesterol in the target membrane enhances the final and dried in a propane flame immediately before use. Flow-cell chambers extent of fusion for Sindbis virus. Cholesterol forms micro- were built by attaching glass coverslips to the quartz microscope slides, with domains, or lipid rafts, when combined with sphingomyelin in channels defined by double-sided tape. The ends of the channels were sealed lipid bilayers (39–42). These microdomains are not required with 5-min epoxy. Fluid was introduced into the channel through holes drilled in the quartz slide at either end of the channel. To form supported for Sindbis virus fusion and may actually inhibit fusion bilayers on the walls of the channel, liposomes were incubated in the cham- (41,43) by reducing lipid mobility (44–49). Here, we find that ber for 5 min and then rinsed away. A second incubation with 20 mg/ml of inclusion of cholesterol in the target membrane increases the PC liposomes for 1 h improved overall experimental reproducibility. interval between binding and lipid mixing at pH values ,5 for A home-built buffer exchanger pumped solutions through the flow cells Sindbis but not for influenza; a result that emphasizes the during observations with the microscope. Virus and acidic buffers were contained in separate syringes and mixed in a tee immediately before injec- complex interactions between lipids and proteins in mem- tion into the flow cell. A variable-speed motor actuated the syringes. Enough brane fusion and may point to subtle differences between the solution was perfused to ensure that ;2.5 channel volumes were injected mechanisms of fusion for type 1 and type 2 viral fusion through the flow cell within ;5s(;20 ml/s) during each experiment. proteins. Sindbis virus will stick to many surfaces. We minimized surface adsorp- tion by constructing the buffer exchange apparatus using PEEK (poly- etheretherketone) tubing and fittings from Upchurch Scientific (Oak Harbor, WA) and disposable syringes (Norm-Ject, Henke Sass Wolf, Tuttlingen, MATERIALS AND METHODS Germany). Clear Tygon tubing was used immediately before the flow-cell entrance to diagnose the presence of air bubbles. Virus and dye labeling Influenza (A, X:31, A Aichi/68, H3N2) grown in fertilized chicken eggs and Lipid mixing hemifusion/fusion assays purified by density-gradient ultracentrifugation was purchased from Charles River Laboratories (Wilmington, MA) and used as provided (2 mg/ml viral For bulk measurements, equal volumes of liposome solution (concentrations protein). as extruded, except for PC which was used at 4 mg/ml) and dye-labeled virus Sindbis was grown in baby hamster kidney cells (BHK-21) cultured by solution (;0.7 3 1012 particles/ml for Sindbis; 0.07 or 0.7 mg/ml viral standard methods in minimal essential media with Earl’s salts containing protein for influenza) were mixed in a fluorescence cuvette. A fluorimeter 10% fetal bovine serum, 5% tryptose phosphate broth, and 2 mM glutamine. (Shimadzu, Kyoto, Japan) detected fluorescence emission from the cuvette Cells were inoculated with Sindbis virus and incubated for 12 h at 37°C. using excitation at 510 nm and emission at 570 nm. After the initial emission

Supernatant was then collected and clarified by low-speed centrifugation. intensity (Io) was recorded, the mixture was acidified by addition of an equal Sindbis virus was purified from the clarified supernatant by ultracentri- volume of an acidic buffer pretitrated to yield the desired final pH. The fugation on a step density gradient followed by a continuous density gradient emission intensity (I) was recorded for 3–5 min, and at the end of the ex- in phosphate-buffered saline (PBS, 10 mM phosphate, 140 mM sodium periment, ;1% vol/vol of 100 mM dodecyl-maltoside was added to dissolve chloride, pH 7.4), containing variable amounts (15–35%) of potassium the samples and yield the unquenched intensity (If)(16,21,22). Dequenching tartrate to adjust the density. Purified Sindbis solutions were adjusted to a was calculated as (I (Io/2))/If and converted into a percentage for plotting. concentration of 2 3 1012 particles/ml as calibrated by BCA Assay (Pierce Single virus particles were observed with a prism-type total internal Biotechnology, Rockford, IL). Sindbis was used immediately or stored at reflection florescence microscope. Samples were illuminated with 8–10 mW –80°C without noticeable loss of fusion capacity (50). laser light (532 nm). Fluorescence emission was collected by a water Both Sindbis and influenza were dye-labeled with the hydrophobic immersion objective lens (PlanApo 603, NA 1.2, Olympus, Melville, NY), fluorescent dye R18 (octadecyl rhodamine B chloride) from Molecular filtered by long-pass filter (HQ545lp, Chroma Technologies, Rockingham, Probes (Invitrogen, Carlsbad, CA). Aliquots of virus (100 ml each, Sindbis at VT), and detected with a CCD camera (Cascade 512B, Photometrics, Roper 2 3 1012 particles/ml, influenza at 0.2 or 2 mg/ml viral protein) were rapidly Scientific, Tucson, AZ) at 10 or 67 frames/s. Custom programs written in mixed at room temperature with 3 ml of dye dissolved in ethanol at 1.4 mM. MATLAB (The MathWorks, Natik, MA) identified individual spots within The dye-virus mix was incubated on ice for 2 h. Virus was purified from movies representing individual virus particles. Intensity time traces were unincorporated dye by gel filtration (NAP 5, G.E. Biosciences, Piscataway, extracted from the movies at those locations by integrating the background- NJ) at room temperature in PBS (for Sindbis) or Hepes-buffered saline subtracted signal from a 7-pixel-diameter circle (1.8 mm in the sample plane)

Biophysical Journal 93(2) 526–538 528 Wessels et al. centered on the identified spots and averaging by the integrated number of upon multiple attempts at identical bulk solution virus-liposome fusion pixels. Virus solutions with low pH were injected into the flow cell while experiments, was ;10% for both viruses in our lab. movies were recorded. For injection into flow cells, virus solution at neutral pH (;0.7 3 1012 particles/ml for Sindbis; 0.07 or 0.7 mg/ml viral protein for influenza) was contained in one syringe and acidic buffer was held in a RESULTS second syringe. When injection was triggered, a motor activated both Bulk solution experiments syringes. The two solutions mixed in a tee (mixing time ,1 s) and then traveled to the entrance of the flow cell. We measured a 1-s delay between Fusion of virus with liposomes in bulk solution has been solution leaving the tee and appearing in the field of view in a flow cell studied with fluorescence dequenching for many years. mounted on the microscope. The virus was thus acidified 1–2 s before it was exposed to the lipid bilayer. Particles then encountered the bilayer at random Before developing a single-particle fusion assay, we con- times after their free diffusion in the flow cell. firmed that our Sindbis and influenza viruses exhibit fusion Different acidification buffers were used depending on the virus and the behavior consistent with this large body of past work. In bulk desired final pH. For Sindbis experiments, 0.1 M Mes/0.2 M Acetic acid/145 assays, samples of virus labeled with self-quenching concen- mM NaCl was added to achieve the lowest final pH of 4.1. For all other final trations of lipophilic, fluorescent dyes are mixed in a fluo- pH values, 100 mM Mes/150 mM NaCl buffer with pH adjusted with NaOH was diluted with variable amounts of TBS to yield a final pH as indicated in rescence cuvette with liposomes. Upon acidification of the the text when mixed with virus samples in PBS. Control experiments at solution in the cuvette, virus particles fuse with liposomes, neutral pH used TBS (pH 7.3). For influenza experiments, the second resulting in dilution of the fluorescent dye and release of the syringe contained 500 mM Mes/145 mM NaCl adjusted to various pH to self-quenching of the fluorescent dye. The intensity increase yield the indicated final pH when mixed with virus in HBS. HBS (pH 7.3) due to the dequenching of most of the lipophilic dyes used in was used for the influenza control experiments. Both bulk and single-virus- particle experiments were performed at room temperature. these bulk fusion experiments (as well as our single-particle The precise values of our ensemble measurements of single-particle assay described below) only reports lipid mixing, but this behavior (see Table 1, and Figs. 1, 2, and 6) were variable over the months of signal has correlated with content mixing in some cases experimentation of this project. Different purifications of Sindbis virus or (21,25,37). different manufacturer lots of influenza virus exhibit different infectivity and The release of the self-quenching of membrane dye during fusion capacity. Different substrates and different manufacturer lots of lipids generate heterogeneity in the properties and defects in the supported bulk fusion experiments leads to an increase in the fluores- bilayers. To minimize the effects of the variability of our samples on the cence emission intensity that reaches a steady value typically ensemble measurements presented here, all data for any given figure were within a few minutes. The emission is scaled to the com- acquired from the same sample, on the same day, on substrates prepared in pletely de-quenched value measured upon addition of de- batch with identical cleaning treatment and using the same supply of lipids. tergent to completely dissolve the vesicular structures in the To estimate the expected variability in the quantitative values from our experimental protocol, we simulated multi-experiment runs all in one day, cuvette. Time courses of typical bulk fusion experiments but instead of scanning parameter space, as was done to generate the data in using our Sindbis virus are displayed as the continuous traces the figures of this article, we repeated one condition in parameter space many in Fig. 1. Similar results are found when using influenza. No times. From these repeated measurements of a single parameter space point, de-quenching from membrane fusion is detected in control we estimate the standard deviation for number of fusions at 44% (Sindbis) experiments where the pH is maintained near neutral (Fig. 1, and 36% (influenza). Standard deviations for the median residence times are 25% (Sindbis) and 21% (influenza). The fact that both Sindbis virus and lowest curve). influenza have similar quantitative variability suggests that these effects are Fig. 2 (solid symbols) displays the pH dependence of the random. The spread in our parameter-space data generally agrees with this final extent of the dequenching signal resulting from fusion variability in regions of parameter space where previous bulk solution for Sindbis and influenza viruses, as deduced from bulk experiments suggest there should be no systematic variation. The main measurements with liposomes. In agreement with previous ensemble effects that we claim in this article (increased fusion at low pH and increased residence time for Sindbis at pH below 5 for cholesterol containing published work, we find that fusion with a physiological target membranes) are effects substantially larger than these random run-run blend of lipids in liposomes (total liver extract) is rapid and variations. The standard deviation of the final dequenching percentages, nearly complete for Sindbis virus for pH ,;6.5, whereas

TABLE 1 Hemifusion/fusion of Sindbis virus with bilayers formed from mixtures of purified lipids Final % dequenching Final % dequenching Residence time single virus: Membrane composition Molar ratio (bulk solution) pH 4.6 (bulk solution) pH 5.5 bilayer pH 4.6 PC/PE 1:1 0 0 0.2 s PC/PE/SM 1:1:1 1 0 0.26 s PC/PE/CH 1:1:1.5 17 2 0.75 s PC/PE/SM/CH 1:1:1:1.5 20 8 0.5 s Liposomes of the indicated mixtures of lipids (CH, cholesterol; SM, sphingomyelin) were used in bulk solution assays or to form supported lipid bilayers for single particle studies. The final extent of dequenching from bulk fusion experiments illustrates the strict cholesterol requirement for fusion with Sindbis virus. The essential nature of sphingomyelin is evident at pH 5.5 but is reduced at pH 4.6. The column of residence times is from single-particle bilayer experiments at pH 4.6. In the absence of cholesterol, there were fewer events in agreement with the lower final dequenching extent in the bulk studies, but residence times could be determined from the few events that did occur. The residence time increased in the presence of cholesterol only for experiments with pH ,5.

Biophysical Journal 93(2) 526–538 Single Virus Bilayer Fusion 529

specific blends of lipids are used to form target membranes (54). The agreement between these bulk studies using virus in our lab and the published work of other labs validates our starting point to design a single-particle assay for viral mem- brane fusion.

Single-virus assay To exploit the advantages afforded by the single-particle experimental approach (55,56), we developed an assay to observe individual Sindbis and influenza virus particles fusing to a supported lipid bilayer. For this assay, we formed a bilayer on a quartz surface within a liquid flow cell. The FIGURE 1 Bulk solution hemifusion/fusion of Sindbis virus with lipo- bilayer was illuminated with total internal reflection of laser somes formed from total liver extract triggered by low-pH conditions. The light, which results in fluorescence emission only from dye- continuous lines show how fluorescence emission from self-quenched dye in labeled virus particles that are less than a few hundred nano- the virus membrane increases after hemifusion/fusion to liposomes dilutes meters from the bilayer. We observed the illuminated region the dye. Buffer is added at time zero to change the pH to the indicated final of the bilayer with a sensitive fluorescence microscope value. Lipid mixing does not occur at neutral pH. The large circles are the accumulated number of dequenching events as a function of time during the capable of single-molecule detection. single-particle bilayer experiment from Fig. 5 B. Similar ensemble kinetics Membrane fusion is triggered by low pH for both Sindbis are observed in the bulk experiments and the single-particle bilayer ex- and influenza viruses. The priming of the particles for fusion periments. upon low-pH exposure is transient and within a few minutes of initial acidification they are rendered incompetent for influenza requires a lower pH of ,6.0 to reach highly efficient fusion. We therefore built a two-chamber buffer exchange fusion with the same type of liposomes (16,20,21,52,53). system to change the pH of the virus solution immediately Table 1, columns 3 and 4, contains results of bulk fusion before it was pumped into the flow cell. Virus was contained experiments where the composition of the target liposome is at neutral pH in one chamber, separate from the low-pH solu- varied. Again consistent with previous work (21), we find an tion in the other chamber. Upon activation, the buffer ex- enhancement of fusion efficiency for Sindbis virus when the changer mixed the solutions in a tee and pumped the mixture target membrane contains both cholesterol and sphingomye- into the flow cell containing the supported lipid bilayer while lin. The data in Table 1 only include a small region of the the flow cell was being observed with the microscope. The parameter space relevant to fusion. For example, it is clear pumping was slow to allow mixing of the solutions in the tee. that the composition of the target membrane determines the The delay between solutions leaving the mixing tee and ar- optimal pH for fusion, as demonstrated by the observation riving in the flow chamber was ;1s(;30 ml in the con- that influenza can fuse to liposomes at neutral pH when necting lines and tee). Virus samples were labeled with self-quenching concen- trations of fluorescent membrane dyes in the same manner as in the bulk fusion studies. After injection of the virus sample into the flow cell, individual particles were detected around the bilayer as their Brownian motion took them into the evanescent light field that extends 100–200 nm beyond the supported bilayer. Individual particles diffused into the illu- minated region and then back into bulk solution in control experiments using neutral pH (pH 7.3). Sindbis and influ- enza virus did not bind to the protein free lipid bilayers at neutral pH. For those few particles that did bind the bilayer, hemifusion/fusion was very rare at neutral pH for either virus. Of the few bound particles, only 3% of Sindbis and 7% of FIGURE 2 Comparison of bulk solution to supported lipid bilayer influenza eventually hemifused/fused when the pH was .7.0. measurements of the pH response of membrane hemifusion/fusion for When the final pH was in the range 4.1–6.5, both viruses Sindbis and influenza viruses. The final extents of dye dequenching from quickly bound to and efficiently hemifused/fused with the bulk solution measurements with liposomes as in Fig. 1 (liver extract) are supported bilayer. We found that 60% of bound Sindbis plotted against the left axis. Plotted on the right axis is the total number of individual dequenching events on a 100 mm 3 100 mm area within 12 min hemifused/fused, whereas 30% of bound influenza hemi- after injection of virus and acidic buffer in supported lipid bilayer ex- fused/fused in the low-pH experiments. (These values are periments (liver extract). determined by averaging the ratio of hemifused/fused

Biophysical Journal 93(2) 526–538 530 Wessels et al. particles to total bound particles for experiments in Fig. 2 dilution of the membrane-incorporated dye as it diffuses into that lie between pH 4.1 and 6.5. We find (hemifused/fused)/ the supported bilayer is reported by sudden dequenching of (total bound) ¼ 0.6 (SD 0.05) for Sindbis and (hemifused/ the dye emission by a factor of ;10. In Fig. 3, A and B, the fused)/(total bound) ¼ 0.30 (SD 0.12) for influenza.) This small intensity increase due to binding of a Sindbis particle is value for influenza efficiency agrees with published reports followed by a larger intensity increase 0.2 s later that is due using the same influenza product from the identical supplier to membrane hemifusion/fusion (see Fig. 3 B, inset). that found ‘‘two-thirds of the viruses were intrinsically fu- Influenza hemifusion/fusion was similar to Sindbis (Fig. 3 sion defective’’ (19). D). Hemifusion/fusion is confirmed by the observation in the Background-subtracted time courses of emission intensity two-dimensional image data of free diffusion of the lipid dye integrated over a 1.7-mm-diameter spot centered on two isotropically into the bilayer away from the site of binding Sindbis virus particles that bind to the bilayer at low pH are after the large dequenching (Fig. 3 A and Supplementary displayed in Fig. 3, B and C. Binding of a virus particle from Material, Movie 1). The motion of the dye in the bilayer was solution leads to a small, localized emission emerging from generally consistent with diffusion using a diffusivity of ;1– the background. In the absence of lipid mixing, these par- 2 mm2/s for all events measured. (Diffusion after the sudden ticles appear as diffraction-limited spots with very low dequenching is discussed in detail below.) In all measure- mobility. The intensity of a bound and unfused virus particle ments described below we confirmed hemifusion/fusion remains localized and stable for minutes (Fig. 3 C), unbind- events by verifying both the sudden dequenching of the ing only in rare cases. integrated intensity and visualization of the subsequent radial Hemifusion/fusion of a bound virus particle leads to lipid spreading of the membrane dye as it diffused into the sup- mixing between the viral membrane and the bilayer. The ported bilayer.

FIGURE 3 Single-particle hemifusion/fusion events. (A) Images extracted from a 10 frames/s movie (Movie S1) of a single Sindbis virus particle labeled with R18 dye hemifusing/fusing with a supported lipid bilayer (liver extract). The pH is 5.5 and virus has been in solution in the flow cell for ;30 s. Time in seconds is indicated under each frame. Individual dye molecules are visible in the later images as they diffuse away from the initial fusion site. The edges of the squares are 13 mm long. The image is uncorrected camera output values that have been false-colored using the jet color table (MATLAB) with the indicated pixel value mapping. (B) Background-subtracted inten- sity time course for the particle in A. The particle binds the supported bilayer at ;32.5 s. The particle remains attached but unfused for less than a third of a second. The interval between binding and hemifusion/fusion is indicated by a rectangle, zoomed in the inset, and denoted as the residence time. The sudden increase in intensity arises from the dequenching of the membrane dye label as it mixes with the supported bilayer after hemifusion/fusion. The dye disperses by diffusion away from the hemifusion/fusion site, leading to the slower intensity decrease. The smooth line connects the data points to guide the eye. (C) Intensity time course for a Sindbis virus particle on a supported lipid bilayer (liver extract) at pH 5.0 that binds but does not hemifuse/fuse. The intensity from virus particles that do not fuse is stable for much longer than is required for the dye to diffuse away after fusion events. (D) Intensity time course for a hemifusing/fusing influenza virus particle on a supported lipid bilayer (liver extract) at pH 4.6. (E) Logarithmic plot of the event from B overlaid with theoretical diffusion curves (see text) for varying diffusion constants, D. D 1.5–2.0 mm2/s fits the data.

Biophysical Journal 93(2) 526–538 Single Virus Bilayer Fusion 531

Dynamics of sudden R18 dequenching due to lipid mixing during individual events Lipids mixed quickly during individual hemifusion/fu- sion events. Using our 10-Hz imaging mode, the sudden de- quenching of dye emission due to lipid mixing during fusion occurred within a single 100-ms time bin for all Sindbis fusion events. Seventy-five percent of influenza events were consis- tent with dequenching within a single 100-ms bin, whereas the remaining 25% extended up to three time bins (300 ms). As no lipid dye remained at the docking site after hemifusion/fusion (with the exception of a few very rare cases described below and in Movie S2), the sudden dequenching indicates that lipid mixing of the membranes occurs in ,100 ms. To further constrain the dynamics of lipid mixing during individual hemifusion/fusion events we increased the data rate to 67 frames/s (our current instrumental limit). Laser illumination intensity was increased by a factor of 3 above the value used in the 10-Hz movies to increase dye emission levels and maintain an acceptable signal/noise ratio. The brighter illumination increased the photobleaching rate. For these measurements, focusing on the short interval between binding and hemifusion/fusion, photobleaching was not a limiting factor. An example of a fusion event recorded at the 67 frames/s rate is shown in Fig. 4 A. Even when using 15- ms time steps, dequenching for this Sindbis virus event FIGURE 4 Measurements of the dynamics of lipid mixing between individual virus particles and a supported bilayer, at 15 ms/frame. (A) The occurred in two frames (Fig. 4 B). The dequenching intervals intensity versus time for a single R18-labeled Sindbis virus particle on a for many events are plotted as a histogram in Fig. 4 B.Allof supported lipid bilayer (liver extract). The particle binds the supported these events dequenched to approximately the same final bilayer, then hemifuses/fuses in less than two 15-ms time steps. (B)A intensity as illustrated in the inset of Fig. 4 B. The events in histogram of dequenching times (10–90%) for 49 of 51 fusion events taken . this graph were derived from 10 movies using five indepen- over 10 movies from five slides (two were 200 ms). Most events occur in two time steps. Some events that are fast enough to be completed within one dently prepared experiments. Only 2 out of the 51 total events time bin will be counted in the group requiring two time bins, because those exceeded the 0.2-s interval plotted here. events may begin toward the end of one time bin and finish during the next Following Liu et al. (29), we note that for fast kinetics the bin. An exponential fit omitting the first time bin yields a time constant of 28 lack of synchronization of fusion events with the edge of a ms. The histogram in the inset displays the maximum intensity after dequenching for the fusion events analyzed in B. The distribution in the inset time bin in the detector reduces the number of events shows that the events used to measure the initial dequenching rate did not observed to transition within one time bin. Dequenching vary by more than a factor of 3–4 in final intensity, although there is some transitions due to fusion that start close to the end of one heterogeneity in the efficiency of particle labeling. detector integration interval may finish during the following integration interval. Such events will thus span two time bins even if the inherent dynamics are faster than one time bin. diffusion of membrane dye into the bilayer occurred a few Such a suppression of the first 15 ms bin in the distribution seconds later from that bright center. In a few cases, a third of dequenching transition times is clear in Fig. 4 B. The hemifusion/fusion event occurred from the same spot (Movie remaining bins of the histogram match a simple exponential S2). We interpret these multiple hemifusion/fusion events as decay with a time constant of 28 ms (or ;2 time bins). Due being caused by aggregates of virus in which different virus to the similarity of the observed dynamics to our exper- particles in the aggregate hemifused/fused at different mo- imental resolution, we conclude that our observation of the ments. It has also been suggested that aggregation may explain lifetime of states leading to lipid mixing during membrane the observation of multiple fusions at single sites for SNARE- fusion in these viral systems remains instrument-limited and driven fusion of liposomes to supported bilayers (29). that 30 ms is an upper bound. Virus particles appeared to undergo multiple fusions in a Diffusion of R18 in the supported bilayer after few rare cases. These viruses would hemifuse/fuse to the sudden dequenching bilayer, leaving behind a smaller, but still bright, center after the initial burst of diffusing dye in the bilayer dispersed and After sudden dequenching of the dye within the virus par- faded. Another sudden dequenching event followed by ticles, we observed that the R18 dye spread in the supported

Biophysical Journal 93(2) 526–538 532 Wessels et al. bilayer uniformly away from the fusion site. The concentra- tion (number of dyes per area), C(r,t), of R18 in a two- dimensional bilayer that spreads from a sudden point source of No molecules of dye deposited at r ¼ 0 and t ¼ 0is r2=4Dt described by Cðr; tÞ¼ðNo=4pDtÞe , where D is the diffusivity of R18 in the bilayer (57). Integration of this concentration over a circle of radius R around the initial fusion site yields a function proportional to the intensity we observe in the time traces (Fig. 3, B and D) in our experiment, IðtÞ¼Ið0Þ½1 eR2=4Dt. This function is plot- ted in Fig. 3 E for R ¼1.2 mm (our experimental parameter), along with the data from Fig. 3 B, as a log-linear plot for values of D ranging from 1 to 3 mm2/s. D 1.5–2.0 mm2/s fits the data well, capturing the nonexponential behavior. This diffusivity agrees with published reports of R18 mobility in bilayers (33), with our measurements of R18 diffusivity in bilayers using single-particle tracking and fluorescence recovery after photobleaching (data not shown), and also with the mobility of many different fluorescent lipid analogs in purified lipid bilayers (58,59). We often observed a delay between the sudden dequench- ing due to initial lipid mixing and the eventual spreading of lipid dye by free diffusion. Such a delay is evident in Fig. 3 B, where a nearly constant intensity is observed for 0.3 s (three time bins) between the sudden dequenching rise and the eventual spread of dye into the bilayer by diffusion. Not FIGURE 5 Time course of single particle-bilayer experiments. (A) Each all fusion events showed this sort of delay; for example, Fig. of the open symbols (dashed line) corresponds to the total number of 3 D does not have a measurable delay between dequenching hemifusions/fusions observed in the preceding 2-min interval for Sindbis and free diffusion. Several fusion events (55% of Sindbis virus interacting with supported lipid bilayers (liver extract) at pH 4.6. Virus events and 44% of influenza events) did not have the delay at was introduced into the flow cell at time zero. The solid line (solid symbols) reports an identical measurement of virus that was exposed to pH 4.6 for the top, whereas the remaining events showed a subdiffusive 6 min before the injection into the flow cell. Sindbis loses its fusion capacity pause after the sudden dequenching. These pauses were of quickly after exposure to acidic conditions. (B) Results from a different variable duration, with the most common delays ;0.2–0.5 s experiment at pH 5.5 (without acid exposure to the virus before injection to (2–5 time bins), but a few pauses extended up to ;1 s. This the flow cell) binned into events within the preceding 10 s illustrates that the delay may indicate that the viral fusion proteins at a site of majority of lipid mixing events takes place within the first minute of the experiment. The large circles in Fig. 1 plot this same data as the cumulative hemifusion/fusion alter the free flow of lipids between the number of hemifusion/fusions as a function of time. viral particle and the target bilayer (60–63).

Fig. 1, the accumulation of dequenching events from the data The ensemble of particles in solution in Fig. 5 B is plotted as large circles along with the bulk Immediately after acidification and injection into the flow solution experiments. Similar overall kinetics is observed in cell, we observed a burst of activity, with virus particles the bulk solution and bilayer experiments, with most fusion binding and hemifusing/fusing with the supported bilayers. being completed in the first minute or two although there is a After continued exposure to acidic conditions for only 1–2 small amount of fusion continuing for several more minutes. min, the frequency of events leading to lipid mixing de- The total number of individual hemifusion/fusion events clined. Sporadic events were detected out to 12 min. By occurring on supported bilayers within 12 min (analogous to recording the time of each fusion event relative to the initial the final extent of dequenching in bulk solution experiments) introduction of virus into the flow cell, we were able to for experiments at varying pH is reported in Fig. 2 (open compare the time course and parameter dependence of fusion symbols). The integrated activity in bilayer experiments in our bilayer assay with bulk fusion assays. As shown in matches the trends observed in the final extent of dequench- Fig. 5 A, almost all of the fusion activity of Sindbis virus was ing for bulk fusion experiments despite the substantial completed within 4 min, in agreement with bulk fusion difference in curvature of vesicles and supported bilayers. studies (21). Fig. 5 B displays the rate of fusion for earlier The decline of the hemifusion/fusion rate is due to both the times, demonstrating that there is a burst of activity within overall depletion of virus from solution due to hemifusion/ the first half-minute of exposure to acidic conditions. In fusion, as well as unfused virus in solution losing its fusion

Biophysical Journal 93(2) 526–538 Single Virus Bilayer Fusion 533 competence after incubation in acidic conditions. In control The surprising exception to the short residence time is experiments where virus was mixed with low pH for 6 min evident in Fig. 6 for Sindbis virus fusing with total liver before being introduced onto the bilayer, fusion was almost extract bilayers below pH 5. In this particular system, the completely eliminated (Fig. 5 A, solid symbols). In control residence time was longer by more than a factor of 10. The experiments at neutral pH, the rate of binding and lipid- only other condition in which longer residence times were mixing events was much lower, although the few individual observed was in the hemifusion/fusion to blends of purified events that did occur could be analyzed for their detailed lipids (Table 1, last column). The inclusion of cholesterol behavior as described in Figs. 2 and 6. and sphingomyelin into mixtures of PC and PE lipids increase the residence time of Sindbis virus by a factor of 2–3 for pH 4.9. The residence time: the interval between membrane binding and membrane fusion for individual virus particles DISCUSSION The single-particle approach allowed us to measure the time Membrane fusion is a rapid, nonequilibrium process, and interval between binding of an individual virus particle to the high-resolution observations of its dynamics will greatly bilayer and the hemifusion/fusion of that particle to the advance our understanding of the mechanics underlying this bilayer (Fig. 3 B, inset, arrow). Measurements of the median important biological phenomenon. Membrane fusion is often of this time interval, which we call the residence time, for described as triggerable by environmental conditions; for ensembles of fusing influenza and Sindbis particles are instance, release is triggered by calcium plotted in Fig. 6 for varying pH. Note that in our assay, viral influx during action potentials, or many viruses fuse mem- priming and activation processes due to acidic exposure are branes upon acidification of endosomes. In actuality, trig- completed before the membrane is engaged by the particles. gering is only a change of the probability (sometimes Therefore, our residence time is not equivalent to the delay dramatically) that membranes will fuse. Even under highly time reported in experiments where influenza fusion proteins promoting conditions, the occurrence of any single fusion (hemagglutinin (HA)) are prebound to a target membrane by event is still stochastically distributed. The single-particle receptor interactions at neutral pH. Those delay times, which approach is well suited to reveal the mechanistic details are longer, include low-pH priming and activation steps (see about protein-mediated membrane fusion because single- Discussion). At pH near 6.9, fusion events were rare, but we particle analysis avoids averaging the behavior of ensembles were able to calculate median residence times from the few of particles and is therefore sensitive to the transient dy- events that were observed. In most cases, hemifusion/fusion namics during membrane fusion. follows quickly after each virus particle binds the bilayer. Here, we describe an in vitro assay for high-precision Particles that hemifuse/fuse spend a median time of only measurements of lipid mixing during early stages of indi- ;0.2 s on the bilayer. A few virions remain stably docked on vidual viruses fusing with supported lipid bilayers in re- the bilayer for half a minute or longer before eventually sponse to low pH. In our assay, an ensemble of virus labeled hemifusing/fusing. with quenched fluorescent dye is acidified and immediately introduced into a flow cell with a protein-free, supported lipid bilayer on its surface. Particles encountered the bilayer by diffusion, so we used a fluorescence microscope to ob- serve the bilayer for up to 12 min (Fig. 5). When particles encountered the bilayer, many bound and hemifused/fused. Lipid mixing commenced around a quarter-second after binding (Fig. 6). The sudden dequenching transitions during individual lipid mixing events were resolution-limited at 30 ms (Figs. 3 and 4). As the ensemble was monitored for the 12-min interval, the rate of individual hemifusion/fusion events decreased, largely due to the loss of fusion capacity after acidification of the virus (Fig. 5). Our assay has repro- duced many results previously found in bulk solution exper- FIGURE 6 Residence times for Sindbis and influenza virus as a function iments, including the pH dependence of fusion for Sindbis of pH. The median residence time (as defined in Fig. 3 B and the text) and influenza, the inactivation of fusion capacity by preex- accumulated from many experiments for Sindbis virus increases sharply by a posure to acidic conditions, and the requirement for choles- factor of 10 at low pH compared to the near-constant residence time for terol and sphingomyelin in Sindbis fusion (37,39–43,45). influenza. Note that most events occur quickly after virus binds to the bilayer. No pH dependence was observed for influenza, but at the lowest pH Our supported lipid bilayer assay differs from the bulk- values, the residence time for Sindbis virus increased by more than a factor solution-based fusion assays in several important aspects. In of three. the bilayer experiments, it is clear that the virus is exposed to

Biophysical Journal 93(2) 526–538 534 Wessels et al. low pH before encountering the target membrane. In bulk can synchronize fusion dynamics for many particles after the fusion experiments, this distinction is difficult to verify. The data have been recorded. In this way, we directly confirm successful crystallization of low pH conformations of trimers that lipid mixing occurs during individual Sindbis virus of type 2 fusion proteins, as are present in Sindbis, required fusion events faster than 30 ms and that hemifusion/fusion the presence of liposomes and lipidlike detergents(64,65). rapidly follows membrane binding. Influenza hemifuses/ This biochemical result suggests that the virus might require fuses with similarly fast kinetics. Lipid mixing is complete, exposure to lipids before the acidification. Our single-particle with no dye remaining at the fusion site after the dispersal by approach allows the sequence of acidification, binding, and diffusion into the bilayer after a few seconds. Niles and hemifusion/fusion to be observed, and demonstrates that acid- Cohen (33) imaged fusion of individual influenza particles ification of virus particles before lipid exposure maintains with planar lipid bilayers with results that are consistent hemifusion/fusion capacity. with our measurements. They observed the initial sudden Criticisms of spontaneous dye transfer between virus and dequenching to be complete on the order of 0.1 s and the dye target membranes independent of membrane fusion (66) are to radially disperse in a few seconds. Niles and Cohen also avoided in our assay. Such spontaneous transfer is easily observed that the overall rate of fusion declined to zero over rejected in our data analysis, as it is much slower than the 3–5 min after injection into the low-pH solution around the dequenching due to fusion (67) and also involves a much bilayer. Our higher resolution allowed us to further constrain smaller percentage of dye than our complete particle fusion the upper limit of the dequenching event to 30 ms and to signals (25,26). determine the intensity trajectory of fusion events for quan- In bulk experiments, fusion typically occurs between virus titative comparison to diffusion theory. particles and highly curved, small liposomes. Supported lipid Diffusion theory accurately described the radial transport bilayers have zero curvature and more closely approximate of the dye in the bilayer away from the initial particle at- the geometry of physiological situations where viruses fuse tachment site after the sudden dequenching due to a lipid to larger structures. Different energetic concerns arise related mixing event (Fig. 3 E). Careful examination of the data for to supported lipid bilayers. Fusion of a virus particle to a the few tenths of a second between the sudden dequenching supported lipid bilayer requires compression of the bilayer to and the eventual diffusive flow revealed subdiffusive lipid accommodate the extra lipids. Also, interactions with the spreading (Fig. 3 B). Other published work examining lipid supporting surface will alter the energy balance between the mixing during membrane fusion of HA cells to red blood virus particle and the target membrane. It is not clear how cells (RBC) or fusion of influenza virus to RBC (32,60,63) relevant any of these considerations are to the in vivo situ- have also found subdiffusive lipid dye mixing. Those experi- ation of in living cells, but the rapid, efficient ments using RBC as the target membrane saw dequenching hemifusion/fusion observed in our experiments suggests that and lipid mixing that continued for minutes. Studies of the these effects are not substantial barriers. fusion of influenza virus with protein-free bilayers found less Our assay only diagnoses hemifusion between the restriction of lipid transfer than the RBC experiments, with supported lipid bilayer and the virus particle. R18 rapidly the lipid dye dispersing at a speed consistent with free dif- flips between the inner and outer leaflet of a labeled bilayer fusion in the bilayer in a matter of seconds (33). These results membrane, and therefore the complete lipid mixing between led to the suggestion that the viral proteins could restrict lipid individual virus particles and the supported bilayer that we flow at a fusion site (61–63). observe is expected for either arrested hemifusion or full Our observation of a brief, subdiffusive pause between fusion. The geometry of the supported bilayer clearly pre- initial lipid dequenching and subsequent diffusive lipid vents the late stages of fusion from progressing naturally, as spread in the bilayer using influenza hemifusing/fusing with there is not room to accommodate the viral capsid between protein free bilayers supports the idea that HA restricts lipid the bilayer and the solid support. It is possible that this flow at a fusion site. That we observed roughly half of restriction will prevent formation of the final fusion pore and hemifusion/fusion events for both Sindbis virus and influ- limit this assay to studies of the initial lipid mixing aspects of enza virus with similar subdiffusive, subsecond pauses protein-mediated membrane fusion. Further experiments will suggests that the restriction of lipid mixing by fusion proteins attempt to confirm full membrane continuity using an aque- occurs for both type1 fusion proteins and type 2 fusion pro- ous content dye or a lipid dye that asymmetrically differen- teins, and thus supports the idea that the mechanisms of tiates between the inner and outer leaflets. Adaptation of our action for type 1 and type 2 fusion proteins are similar single-virus-particle assay to fusion with immobilized lipo- (64,65). somes (our unpublished results and Yoon et al. (68)) will also As a result of many different types of experimentation, a address the issue of hemifusion versus full fusion in this unified model of the sequence of events underlying influenza- assay. HA-mediated membrane fusion is emerging (13,14,69,70). Imaging individual events allows us to measure kinetic At neutral pH, HA on the influenza particle bind their re- features of the rapid transitions during the hemifusion/fusion ceptor, sialic acid, which is present on glangliosides and pro- process. By recording time trajectories for each particle we teins in a targeted cell membrane. After acidification of the

Biophysical Journal 93(2) 526–538 Single Virus Bilayer Fusion 535 medium around this receptor-HA complex, HA molecules iments, virus in our assay is exposed to low pH before en- undergo conformational rearrangement into a primed, or countering the membrane. activated, state. This activated state is metastable, and after We measured the time interval between bilayer binding tens of seconds to a few minutes, in the absence of membrane and hemifusion/fusion for many individual virus particles. In fusion, HA decays to a dead-end state incapable of catalyzing Fig. 3 B, we denote this interval as the residence time. Our fusion. It is expected that activated HA trimers must ag- residence time differs from the previously described delay gregate until a threshold density (estimated to be ;3–8 mol- time for prebound experiments, because in our assay we ecules per fusion site (71,72)) is achieved that is capable of expect that low-pH priming reactions occur while the virus is catalyzing fusion through further conformational changes. In in solution. Under most conditions, for both viruses, we find the event that the rapid fusion transition is triggered, a number that the residence time is around a quarter-second. Measure- of transient intermediate structures form, including small ment of the residence time for Sindbis virus interacting with pores capable of conducting ionic currents, hemifused stalks liposomes has previously been attempted using coflotation permitting lipid mixing between the membranes, and full during density-gradient centrifugation (21). Our results quali- fusion pore formation capable of transferring larger mole- tatively agree with these earlier experiments that found that cules between the two fusing compartments (10,73). not all virus binds to liposome, and additionally not all of the Different assays applied to influenza-HA-mediated mem- bound virus will fuse with the bilayer. However, our assay brane fusion are sensitive to different aspects of this overall measured a much shorter delay between the binding and lipid progression. The most common assays detect fluorescence mixing than was determined with the bulk assay. The in- dequenching or ionic current when whole influenza virus herent delay and insensitivity to weak binding of the cen- (18,33,60,74), mammalian cells engineered to express HA on trifugation measurements may account for these differences. their surface (HA cells) (32,63,74–76), or purified HA recon- The delay time observed in prebound experiments is stituted into liposomes (77) fuse with target membranes in longer than our residence time. The prebound virus exper- liposomes, planar lipid bilayers, or, commonly, RBCs. Experi- iments measure the additional delays of protonation and mental configurations detect fusion of either in bulk solution priming after acidification that in our assay occur before or spatially resolved individual viruses or individual cells. particles encounter the targeted membrane. In all cases, the In assays where virus is not prebound to a target dynamic transition from a primed and membrane-engaged membrane, the diffusional search for a virus to encounter a fusion complex to lipid mixing is rapid and remains tem- target membrane typically limits fusion rates. Many assays porally unresolved by any experiment. We emphasize that that use neutral-pH prebinding of virus or HA-expressing our assay primes virus particles before they are bound to the cells to a target membrane via receptor interactions have membrane, the opposite of the situation in assays where virus found a delay between acidification and fusion, often called is prebound to the target membrane at neutral pH by receptor the lag time. The lag time is found to be ;1–5 s by stopped- interactions. flow measurements (74) or single fusion imaging of whole The residence time for Sindbis virus increased in the influenza virus fusing with RBCs (60) but lengthens to the presence of cholesterol at the lowest pH values tested. The range of 20 s to several minutes for HA cells fusing with role of cholesterol and sphingomyelin in virus entry is RBCs (18,60,63,74,76). A series of experiments varying the complex (41). The combination of cholesterol and sphingo- levels of HA expression in transfected cell lines found in sufficient concentrations in membranes is known shorter lag times as the concentration of functional HA in the to cluster into microdomains, or lipid rafts (78,79), that some membrane increased. These results led to the conclusion that viruses including and human immunodeficiency virus at least some part of the lag time is due to diffusion and (HIV) require in the target membrane for infection (80,81). oligomerization of primed fusion proteins to a sufficient den- Membrane fusion by influenza does not require cholesterol sity to catalyze the fusion transition. The lag time depends in the target membrane (20), although cholesterol in the viral upon the composition of the target membrane (52,74), the membrane has recently been implicated to be important for temperature (18,63), and pH (74), suggesting that the lag time HA trimerization (82). We did not observe any cholesterol may be a convolution of several different kinetic processes. dependent change in the residence time of influenza compa- In our assay, virus is not prebound to the target bilayer. For rable to what we observed with Sindbis. influenza, the supported bilayer assay is easily extendable to Fusion by Sindbis and other alphaviruses strictly requires allow prebinding by incorporating its receptor molecule, cholesterol and sphingomyelin in the target membrane sialic acid on glycolipids, into the bilayer (18,33,34,36,75). (21,83–85), but lipid raft formation is not relevant to this In contrast, Sindbis cannot be prebound because the relevant property (43). Rather, the 3b-OH group on cholesterol has receptor molecule is unconfirmed. (In the event that the been shown to be essential for low-pH binding to membranes receptor molecule is identified, it could likely be incorpo- (85), possibly by interacting with the alphavirus fusion pro- rated into a single virus bilayer fusion assay.) To allow direct tein E1 to stabilize an uncharacterized intermediate confor- comparison to Sindbis virus, we did not include influenza mation that is essential for membrane binding (42). Any sterol receptor in the bilayers. Thus, unlike the prebound exper- with the 3b-OH group substitutes for cholesterol, and further-

Biophysical Journal 93(2) 526–538 536 Wessels et al. more, a point mutation in E1 can eliminate this requirement, Ridge Associated Universities, and a Career Award at the Scientific presumably by stabilizing the necessary intermediates without Interface from the Burroughs Wellcome fund. the sterol involvement (86,87). Although the 3b-OH group plays a specific role in binding, a fusogenic role for choles- REFERENCES terol cannot be ruled out (44,88). Studies around pH 5.5 have found that small amounts (a 1. Chen, E. H., and E. N. Olson. 2005. Unveiling the mechanisms of cell- few percent) of sphingomyelin are required in the target cell fusion. Science. 308:369–373. membrane for fusion, but not for binding of alphaviruses 2. Chernomordik, L. V., and M. M. Kozlov. 2005. Membrane hemi- fusion: crossing a chasm in two leaps. Cell. 123:375–382. (89). It is also postulated that sphingomyelin interacts with 3. Cohen, F. S., and G. B. Melikyan. 2004. 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